Identification of three different truncating mutations in cytochrome P4501B1 (CYP1B1) as the principal cause of primary congenital glaucoma (Buphthalmos) in families linked to the GLC3A locus on chromosome 2p21
Identification of three different truncating mutations in cytochrome P4501B1 ( CYP1B1 ) as the principal cause of primary congenital glaucoma (Buphthalmos) in families linked to the GLC3A locus on chromosome 2p21IvayloStoilov, A. NurtenAkarsu and MansoorSarfarazi*
Surgical Research Center, Department of Surgery, University of Connecticut Health Center,Farmington, CT,USA
Received January 17, 1997;Revised and Accepted January 31, 1997
Primary congenital glaucoma (Buphthalmos) is an autosomal recessive eye disorder, postulated to result from developmental defects in the anterior eye segment. Previously, we reported two chromosomal locations for this condition on 2p21 (GLC3A) and 1p36 (GLC3B) respectively. In this study, heritable mutations of human cytochrome P4501B1 gene (CYP1B1) in affected individuals of five well-characterized families linked to the GLC3A locus are described.CYP1B1gene has previously been mapped within the GLC3A candidate region and its expression in the trabecular meshwork cells has been demonstrated in this study. Three different homozygous mutations were identified and characterized: a 13 bp deletion in exon III; an insertion of a single cytosine base in exon II; and a larger deletion affecting the 5' end of exon III and the adjacent intronic region. All of these are frameshift mutations that are predicted to remove domains essential for the function of theCYP1B1protein. Therefore, it is expected that all these mutations result in functional null alleles. The mutations detected in the affected members of these families were not present in 470 chromosomes from randomly selected normal individuals, thus strongly suggesting thatCYP1B1 is the gene for the GLC3A locus on 2p21. The results are discussed in the context of the earlier hypothesis that `drug-metabolizing' enzymes might modulate the processes of growth and differentiation by controlling the steady-state-levels of oxygenated growth-effector molecules.
Glaucomas are a heterogeneous group of eye disorders, accounting for 15% of blindness worldwide (1 ). A common feature among these conditions is the typical glaucomatous atrophy of the optic nerve that ultimately leads to progressive visual field loss and total blindness (1 ). So far, our knowledge of the primary events capable of triggering optic atrophy and the true nature of underlying pathogenic mechanisms is still limited. A promising avenue of investigation opened when families segregating in Mendelian fashion were reported for different types of glaucoma (2 -9 ). The identification of such families made it possible to search for the glaucoma putative genes by utilizing a general method of genetic linkage analysis, positional mapping and positional cloning. To date, four different glaucoma loci have been assigned to the human genome: the juvenile-onset primary open angle glaucoma (GLC1A) mapped to 1q21-q31 (2 -6 ); one form of adult-onset primary open angle glaucoma (GLC1B) mapped to 2cen-q13 (7 ); the first locus (GLC3A) for primary congenital glaucoma (Buphthalmos) mapped to 2p21 (8 ); and the second locus (GLC3B) mapped to the 1p36 region (9 ). However, as yet, no mutations have been described for any of these four loci. Herein, we report the first mutation in families with primary congenital glaucoma linked to the GLC3A locus on 2p21 and describe the identification of humanCYP1B1 as the gene responsible for this pediatric condition.
Buphthalmos or primary congenital/infantile glaucoma (gene symbol: GLC3) is usually diagnosed at birth or shortly thereafter. Classical manifestations include tearing, photophobia, and blepharospam that is often supplemented by clouding of the cornea (10 ). These symptoms occur secondary to the corneal irritation that accompanies corneal epithelial edema caused by elevated intraocular pressure. Autosomal recessive mode of inheritance is observed in most of the reported familial cases (8 -9 ,11 -13 ). The incidence of the disease is estimated to be 1:2000 in the Middle East and 1:10 000 in the western countries (14 ,15 ). Maldevelopment of the anterior segment angle or goniodysgenesis are the hallmark of congenital glaucoma (16 ). The pathogenesis of this condition, however, has remained enigmatic. Among the proposed pathogenetic mechanisms are a membrane covering the anterior chamber angle and blocking the aqueous humor outflow (17 ); abnormal cleavage of the mesoderm during the formation of the anterior chamber angle (18 ); inhibited posterior migration of the ciliary body and iris root (19 ); and developmental arrest of the anterior chamber angle structures derived from the neural crest cells (20 ). Accordingly, the identification of the mutated gene(s) is expected to provide a much needed guideline in exploring the different pathogenetic concepts. Therefore, the aim of our study was to identify the gene causing primary congenital glaucoma by genetic linkage analysis and positional cloning.
After excluding many genes and candidate chromosomal regions (21 ), we embarked on a random screening of the genome and were able to identify two loci for primary congenital glaucoma; GLC3A and GLC3B were mapped to chromosome 2p21 (8 ) and 1p36 (9 ) respectively. We also provided evidence for the existence of at least one more unmapped locus for this condition (8 -9 ). The GLC3A locus on 2p21 has recently been confirmed in another panel of 25 families from Saudi Arabia (22 ). Critical recombination events initially placed the GLC3A locus within an 8 cM interval that was flanked by markers D2S1325 telomerically and D2S1356 centromerically (8 ).Inspection of the smallest conserved segment of homozygosity in the affected members of our consanguineous families placed the disease locus telomerically to marker D2S1346, substantially narrowing the GLC3A candidate region. A recently reported recombination event with D2S2186 (22 ) has reduced the GLC3A critical candidate region to ~2.5 cM that is flanked by markers D2S2186 and D2S1346 (Fig.1 ).
Three genes namely, the non-erythrocytic form of [beta]-spectrin or [beta]-fodrin (SPTBN1), a guanine nucleotide exchange factor for Ras (hSOS1), and interferon-inducible double-stranded RNA-dependent protein kinase (PRKR) have previously been mapped to the 2p21 region (23 -29 ) and, therefore, were implicated as possible candidate genes for this condition (8 ). In order to refine their positions, we screened these genes against the GeneBridge 4 Radiation Hybrid (RH) panel and mapped them relative to the Whitehead RH framework map (30 ). Screening of the RH panel was carried out by gene specific polymerase chain reaction (PCR) assays. Preferably, the intronic or 3' untranslated sequences were assayed to prevent crossamplification of the hamster DNA background. The following map positions (Fig.1 ) were established for these genes:SPTBN1 mapped 1.51 centiRay (cR) from marker WI-4077 (LOD>3.0; not shown);hSOS1 positioned 1.51 cR from marker WI-10326 (LOD>3.0); andPRKR was placed 2.3 cR from D2S177 (LOD>3.0). Therefore,SPTBN1 maps centromerically to D2S1356 (Fig.1 ) and, thus is excluded as a candidate gene for GLC3A. Inspection of the contig map (30 ,31 ) harboring the GLC3A locus revealed that marker WI-7936 maps very closely to D2S177. This sequence tagged site (STS) corresponds to the gene for human cytochrome P4501B1 (CYP1B1,32 ; Fig.1 ). A fifth gene encoding 9G8 splicing factor (SFRS7) was identified when a BLAST search determined that the expressed sequence tagged (EST) marker TIGR-A004S39 has been derived from the 3' untranslated region of this gene (33 ). This EST has already been mapped next to marker D2S177 on the chromosome 2 RH map (30 -31 ).
Because of the limited understanding of the primary defect in congenital glaucoma, it was difficult to exclude any of the genes mentioned above, based on the physiologic functions of their corresponding proteins. Therefore, they were considered as potential candidate genes for primary congenital glaucoma and their coding sequences were screened for mutations by a direct sequencing method. Total RNA was prepared from human skin fibroblast cultures previously established from affected individuals. cDNA fragments for sequencing were generated by reverse transcription PCR amplification. Initially, we screened the entire coding sequences of thehSOS1 andPRKR genes. When no sequence variants were observed for both genes, we proceeded with mutation screening of theCYP1B1 gene. As a result of this screening, we first detected a 13 bp homozygous deletion (Fam. 26; affected individual 10) that removed nucleotides 1410 to 1422 (i.e., GAGTGCAGGCAGA) from the coding sequences (i.e., exon III) of theCYP1B1 gene (32 ). This mutation resulted in a frameshift that truncated the open reading frame by creating a premature stop codon (TGA), 203 bp downstream of this deletion (or 68 amino acids after the last original amino acid Thr-354). In order to develop an assay for genomic DNA screening, we determined the intron/exon junctions of this gene. We concluded that theCYP1B1 contains three exons and two introns (Fig.2 ). The entire coding sequence of the gene is contained inexons two and three. The genomic structure ofCYP1B1 determined in this study is in agreement with the result published shortly before submission of this report (34 ). The presence of the 13 bp exonic deletion in family 26 and its cosegregation with disease phenotype was confirmed by acrylamide gel electrophoresis of a 124 bp PCR fragment that harbored the deleted region (Fig.3 A). The same 13 bp deletion was also detected and subsequently confirmed to segregate with the disease phenotype in one more family (Fam. 17; Fig.3 A). The latter is an interesting pedigree in which the offspring are the result of a consanguineous marriage between an affected father and a normal mother. These type of pedigrees are sometimes cited as an example of a pseudo-dominant inheritance. However, as shown for this pedigree (Fig.3 A; Fam. 17: DNA was unavailable from one affected and three more normal offspring), the father is homozygous for the 13 bp deletion while his wife is heterozygous for the same deletion. Therefore, the two affected and normal offspring have inherited a single copy of this deletion from their father alone, while the affected offspring, in addition, inherited a 13 bp deletion from his mother. A second mutation was observed in another two families (Fam. 10 and 11) who exhibited a homozygous insertion of an extra cytosine base in a stretch of six cytosines normally located between nucleotide positions 1209 and 1214 in exon II (Figs2 and3 B). This also proved to be a frameshift mutation that created a premature stop codon (TGA), 106 bp downstream from the site of this insertion (or 36 amino acids downstream from the original amino acid Pro-289). Furthermore, a third mutation was detected in another consanguineous family (Fam. 15; Fig.3 C). This is a much larger deletion that starts in intron II and removes a certain portion of coding sequences of exon III that extends behind the above-mentioned 13 bp deletion (Fig.2 and Fig.3 C). Although, we have not determined the exact size of this deletion as yet, it is obvious from PCR amplification pattern that the 5' end of exon III and the adjacent intronic region are definitely deleted, but the 3' end of exon III has remained intact (Fig.3 C). As the 3' splice acceptor site of intron II has been deleted, this mutation is expected to interfere with the normal splicing of theCYP1B1 gene, resulting in synthesis of either truncated protein, or null allele. Analysis of 470 chromosomes from randomly selected normal individuals (330 Turkish and 140 other Caucasians) failed to detect the presence of the three mutant alleles described above, making it less likely that these sequence variants represent rare polymorphisms. As these mutations were only observed in 14 affected subjects in the homozygous state but not in the normal members of a total of five families (four are consanguineous) and, as the normal population from which these families are ascertained did not carry these mutations, this strongly suggests thatCYP1B1 is the gene for the GLC3A locus on 2p21.
Low levels of constitutiveCYP1B1 mRNA have been detected in 15 different tissues (32 ). By reverse transcription and quantitative PCR amplification, we determined thatCYP1B1 is also expressed in the trabecular meshwork, the very primary tissue that is considered to be essentially affected in congenital glaucoma (Fig.4 , ref.35 ).
The families used for identification of the chromosomal locations of this condition have been described in detail elsewhere (8 ,9 ). Briefly, the phenotype observed in these families was characterized by onset during the first months of life, bilateral involvement of the eyes, an autosomal recessive pattern of inheritance and no association with other ocular or systemic anomalies. Cytogenetic analysis revealed normal karyotypes in 24 affected individuals originally used for linkage analysis (8 ,9 ,13 ).
Radiation hybrid mapping of the GLC3A critical candidate region was performed by first designing a series of oligonucleotide primers and establishing the PCR conditions that specifically amplify a portion of a particular gene. In each instance, PCR amplification of the targeted fragment was observed by agarose gel electrophoresis when human genomic DNA was used as template, but not with rodent DNA template. We then scored for the presence or absence of the particular PCR products in 91 radiation hybrid DNA samples from the GeneBridge 4 Radiation Hybrid panel purchased from Research Genetics, Inc. The results of RH screening were reported as a vector of 91 `0's, `1's, and `2's (original MIT order). Each digit corresponds to one of the cell lines with `0' and `1', representing negative and positive PCR assays respectively. A `2' indicates that the assays were contradictory between two or more replicate experiments. Statistical analysis of the RH data was carried out on the mapping server at Whitehead Institute for Genome Research where a detailed map information can be obtained from (http://www-genome.wi.mit.edu/ ).
Monolayers of human skin fibroblasts have been maintained at 37oC in CO2 incubation, in media MEM supplemented with 10% Fetal Bovine Serum and antibiotics. For total RNA isolation, preconfluent cells from 60 mm dish were treated with 2 ml TRIzol reagent (Gibco-BRL). Next, 400 µl chloroform was added and the aqueous phase was recovered by centrifugation. RNA was precipitated with 1 ml isopropanol, washed with 75% ethanol and diluted in 20 µl DEPC treated water. First strand synthesis was primed from 10 µl of the RNA sample with 50 ng random hexameres. Reaction was carried out in 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 0.5 mM from each dNTP, 0.01 M DTT, and 200 U SuperScript II RT (GIBCO) in total volume of 20 µl for 1 h at 42oC. The coding sequence of theCYP1B1 gene was amplified with the cDNA-based primer sets: CYP1 (F 5'-GGTTCCTGTTGACGTCTTG-3'; R 5'-CTTCCAGTGCTCCGAGTAG-3'); CYP2 (F 5'-GTGGTGCTGAATGGCGAG-3'; R 5'-TACTGCAGCCAGGGCATC-3'); CYP3 (F 5'-GTGGCCAACGTCATGAGTG-3';R 5'-TCATAAAGGAAGGCCAGGA- C-3'), and CYP4 (F 5'-AGACTCGAGTGCAGGCAG-3'; R 5'-TCCTCATCTCCGAAGATGGT-3').
Approximately 2 µl from the cDNA sample were subjected to PCR amplification with 20 pmol from each gene specific primer in a total volume of 50 µl that consisted of 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 0.5-2.0 mM MgCl2, 0.2 mM from each dNTP, and 0.5 U recombinantTaqPolymerase (Gibco-BRL). Amplification conditions consisted of 1 min initial denaturation at 94oC followed by 35 cycles of denaturation at 94oC for 1 min, annealing at 53oC for 1 min, and final extension at 72oC for 1 min. PCR was carried out on a GeneAmp 9600 thermocycler (Perkin Elmer). The amplified PCR fragments were purified directly or from agarose gels with Wizard PCR preps DNA purification system (Promega). Dye terminator sequencing withTaqPolymerase FS was performed on an ABI-373 sequencer (Perkin Elmer).
In order to recover the genomic region containing theCYP1B1coding sequence, we used the cDNA based primer sets CYP1-4 in long range PCR amplification with eLONGase enzyme mix (Gibco-BRL) according to the protocol supplied by the manufacturer. Total yeast DNA prepared from the strain containing YAC 806-F-8 (30 ,31 ; Research Genetics) served as a template. Intron/exon junctions in theCYP1B1 gene were identified by comparing the sequences of the amplified fragments to the reference cDNA sequence. For mutation screening, three primer sets were assembled for amplification of theCYP1B1 coding sequence from genomic DNA. Primer CYP1-F was paired with the intronic primer 5'-CCTCCCAGAGGCTTTACCT-3' for amplification of coding sequences of exon II (1.6 kb fragment). For amplification of the coding region located in exon III, intronic primer 5'-TAAGAATTTTGCTCACTTGC-3' was paired with primer CYP4-R (693 bp fragment product). PCR was performed in a total volume of 100 µl that consisted of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM from each dNTP, 40 pmol from each primer, and 2 U AmpliTaq Polymerase (Perkin Elmer). Amplification conditions consisted of 1 min initial denaturation at 94oC followed by 35 cycles of denaturation at 94oC for 15 s, annealing at 55oC for 15 s, and extension of 15 s at 72oC. A 134 bp fragment containing the 3'-end of theCYP1B1 coding sequence was amplified with primers 5'- TCAATGTCACTCTCAGAGAG-3' and CYP4-R.
For rapid mutation screening, a 124 bp fragment containing the 13 bp deletion was amplified from genomic DNA with primers: 5'-CAAACAGGTATCCTGATGTG-3' and CYP3-R. PCR was performed in a total volume of 50 µl that consisted of 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.0 mM MgCl2, 0.2 mM from each dNTP, 20 pmol from each primer, and 0.5 U recombinantTaqPolymerase (Gibco-BRL). Amplification conditions consisted of 1 min initial denaturation at 94oC followed by 35 cycles of denaturation at 94oC for 15 s and annealing at 57.5oC (which was decreased to 53oC at 0.5oC increments during the first 10 cycles) for 15 s. The PCR products were analyzed on acrylamide minigels consisting of 5% Acrylamide/Bis solution (19:1), 15% urea, and 1* TBE. Electrophoresis was carried out in a minigel (70/80/1.0 mm) apparatus (Mini-Protean II, BioRad) with 1* TBE running buffer at 100 V for 45 min. Gels were stained with ethidium bromide. This assay can also detect homozygotes for the large undetermined size deletion as observed in pedigree 15. The mutation detected in exon II was amplified from genomic DNA with primers: 5'-GACAAGTTCTTGAGGCACTGC-3' and 5'-ACGTTCTCCAAATCCAGCC-3'. PCR was performed in a total volume of 50 µl that consisted of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.75 mM MgCl2, 0.2 mM from each dNTP, 20 pmol from each primer, and 0.5 U AmpliTaq Polymerase (Perkin Elmer). Amplification conditions consisted of 1 min initial denaturation at 94oC followed by 35 cycles of denaturation at 94oC for 15 s and annealing at 55oC for 15 s. The amplified fragments were electrophoresed on sequencing type polyacrylamide gel (40/40/0.4 cm) under denaturing conditions. The gel consisted of 7% Acrylamide/Bis solution (19:1), 7 M urea, and 1* TBE. Electrophoresis was carried with 1* TBE running buffer at 60 Watts for 3 h. Gels were visualized by silver staining.
We applied the PCR MIMIC protocol (Clontech) to study the expression of theCYP1B1gene in the trabecular meshwork cells. In this experiment known dilutions of nonhomologous DNA fragment acting as an internal control (PCR MIMICs) are coamplified with constant amounts of sample target cDNA. The amount of target cDNA can be ascertained by determining how much PCR MIMIC is required to produce equal molar quantities of target and MIMIC PCR products. For quantitation of the human glyceraldehyde 3-phosphate dehydrogenase (G3PDH) PCR MIMIC construct and primers were purchased from Clontech, USA. ForCYP1B1quantitation, PCR MIMIC with primer templates for primer pair CYP3 was constructed according the protocol provided by the manufacturer.Monolayers of trabecular meshwork cells have been maintained at 37oC in CO2 incubator, in high glucose DMEM (4500 mg/ml) supplemented with 10% Fetal Bovine Serum, L-glutamine, sodium pyruvate, and antibiotics. Total RNA was prepared as described above. The concentration of the RNA in the sample was determined by spectrophotometry. First strand synthesis was primed with 50 ng random primers from 4 µg total RNA. Two microlitres from the cDNA sample and 2 µl from the 10-fold serial dilutions of the PCR MIMIC were subjected to PCR amplification with 20 µM from each gene primer in a total volume of 50 µl that consisted of 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM from each dNTP, and 2.0 U recombinantTaqPolymerase (Gibco-BRL). Amplification conditions consisted of 1 min initial denaturation at 94oC followed by 30 cycles of denaturation at 94oC for 45 s, annealing at 53oC for 45 s (60oC for G3PDH), and 2 min extension at 72oC. PCR was carried out on the GeneAmp 9600 thermocycler (Perkin Elmer). Five µl from the PCR reaction were then resolved on 2.0 % agarose gel and stained with ethidium bromide.
We are indebted to our patients and their families who participated in this study. We would like to thank Professor M. E. Turacli and Dr S. G. Aktan of the Department of Ophthalmology, Ankara University, Turkey for referral of families and Dr M. Ozguc, Director of (TUBITAK) DNA/Cell Bank and Gene Research Laboratory of Hacettepe University, Ankara, Turkey for facilitating fibroblast skin culture from affected patients and partial DNA extraction. Human trabecular meshwork cells culture was kindly provided by Dr Mary Wirtz, Casey Eye Institute, Oregon, USA. This work was supported by grants from the National Eye Institute (EY-11095) and the University of Connecticut General Clinical Research Center (M01-RR-06192). We would also like to thank the International Glaucoma Association for their continuous help and financial support. The DNA sequencing were carried out at the UCHC Molecular Core Facility using an automated ABI-373 system.
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